1. 2. IRCSS, Institute of Neurological Sciences, Bologna, Italy
Skin nerve phosphorylated alpha-synuclein deposits in idiopathic REM sleep behavior disorder
Elena Antelmi,1,2 V. Donadio,2 A. Incensi,2 G. Plazzi,1,2 R. Liguori1,2
1. Department of Biomedical and Neuromotor Sciences, Alma Mater Studiorum, University of Bologna, Bologna, Italy.
2. IRCSS, Institute of Neurological Sciences, Bologna, Italy

3. iRBD 15 yrs 90% α-Syn mediated neurodegenerative disease
- Autonomic symptoms
Several yrs before pheno-conversion
Postuma et al, 2015
- Olfaction, color vision
5 yrs before pheno-conversin
Postuma et al,
Neuroimaging findings:
SPECT DAT-Scan
Transcranial sonography
Structural neuroimaging2011
15 yrs
90%
α-Syn mediated neurodegenerative disease
iRBD
Time-window to observe pre-clinical synucleinopaties and to test potential markers of impending neurodegeneration
Time-window to apply neuroprotective treatments/approches

4. In iRBD: from Tolosa and Vilas, Brain 2015 p-α-synuclein deposits
8/9 (89%) patients with iRBD
vs 8/12 (67%) patients with PD
Vilas et al, Lancet Neurol 2016
p-α-synuclein deposits in 4/17 patients with iRBD, 1/19 patients with PD and none of the HC
Sprenger et al, Neurology 2015
from Tolosa and Vilas, Brain 2015

5. In PD patients, at skin biopsy, p-α-syn in vivo has been found
In 20% (# 2/10) of iPD patients at the chest vs none at the leg site (Miki et al, 2010);
In 51% (# 16/31) of iPD patients vs none of of the HC (11/31 at the back, 6/31 proximal leg, 4/31 distal leg, 5/31 finger) (Doppler et al, 2014);
In none (# 0/6) of iPD patients (samples from distal legs) (Navarro-Otano et al, 2014);
In 100% (# 21) of iPD patients at cervical site and in 52 and 24% of iPD at thigh and leg sites, respectively and in none of the patients with other form of parkinsonism (# 20) and none of the HC (# 30)(Donadio et al, 2014);
In 67% (# 20/30) of iPD and of MSA vs none of the HC and of patients with tauopathy, proximal-distal gradient (Doppler et al, 2015);
In 100% (# 10) of iPD patients vs none of MSA patients at the ventral forearm (Zange et al, 2015)
In 100% (# 16) of iPD patients at cervical site and 75 and 31% at the tight and leg sites, respectively vs 100% (# 14) of PAF patients in all sites and vs none of the HC (# 15) (Donadio et al, 2016).

6. Easy to obtain/perform/not invasive
Aim of the study
To search for misfolded p-α-synuclein deposits in skin biopsy samples as a precocious bio-marker of impending neurodegeneration in patients with iRBD
Ideal Biomarker
100% sensitivity
100% specificity
Not expensive
Easy to obtain/perform/not invasive
Long lead-time

7. Aim of the study Methods
To search for misfolded p-α-synuclein deposits in skin biopsy samples as a precocious bio-marker of impending neurodegeneration in patients with iRBD
Methods
Inclusion criteria
Patients with iRBD (vs HC and iPD)
Positive chronic history of «dream-enacted behaviors» plus PSG-detection of RSWA and of at list one clear RBD episode
Absence of motor impairement at the clinical examination
Absence of cognitive signs at the neuropsychological investigation
Normal Brain MRI
No assumption of drugs that have been related with RBD (i.e. anti-depressant, beta-blockers)

8. Methods investigations
History-tacking; neurological examination and smell test (sniffing stick test kit)
Questionnaires: ESS, PSQI, BDI, STAI, BIS-11
Optical coherence tomography, pupillometry, color vision assessment
SCOPA-AUT questionnaire
Full-night video-PSG (conventional EEG, bilateral EOG, submentalis, bilateral flexor digitorum superficialis of the arms, bilateral anterior tibialis EMG, respiratory parameters, electrocardiogram, infra-red video)
Brain MRI
SPECT DatScan
Nerve conduction velocities study
MMSE and neuropsychological tests
Blood test; CSF analysis for neurodegenerative biomarkers
3 mm punch biopsy at the proximal sites (bilateral paravertebral C8 level) and distally (bilateral distal leg; 10 cm above the lateral malleolus), two different samples (3 to 4 cm apart) were taken

9. minimally invasive requiring no suture
3 mm punch biopsies
minimally invasive requiring no suture
minor discomfort for the patient
1. Ten µm skin sections double-immunostained overnight with rabbit monoclonal phosphorylated α-synuclein at Ser129 (p-syn; 1:500, Abcam, ab-51253) and mouse pan-neuronal marker protein gene product (PGP) (1:750; Abcam, ab72911).
2. Sections were then washed and incubated for one hour after adding secondary antibodies (i.e. mouse or rabbit Alexa Fluor 488 and rabbit or mouse cyanine dye fluorophores 3.18; 1:200 or 1:400, Jackson ImmunoResearch, for r-AlexaFluor488 and for m-cyanine 3).
3. Sections were finally analyzed under a Nikon confocal microscopy (Eclipse Ti A1). Intraneural p-α-syn staining was demonstrated by a colocalization with PGP.
Two authors experienced in immunofluorescence analysis carried out the analysis, blind to the clinical diagnosis.

11. Antelmi et al, Neurology 2017
12 iRBD vs 50 age and sex-matched HC
Antelmi et al, Neurology 2017

13. skin biopsy:
# 10/18 iRBD (sensitivity 55.6%)
# 20/25 early PD (sensitivity 80%)
none of the HC (specificity 100%)
Percentage of samples positive for p-α-syn correlated negatively with the DAT binding at the FP-CIT-PET, olfactory function and positively with total LR for RBD to present prodromal PD

14. * San Raffaele Hospital, Milan
yes
* San Raffaele Hospital, Milan

15. Thank you